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Wednesday, 26 February 2014

Researchers Generate New Neurons in Brains, Spinal Cords of Living Adult Mammals

Wednesday, 26 February 2014

UT Southwestern Medical Center researchers created new nerve cells in the brains and spinal cords of living mammals without the need for stem cell transplants to replenish lost cells.

This image shows Dr. Chun-Li Zhang,
left,

assistant professor of molecular
biology; and

Dr. Zhida Su, a UT Southwestern
visiting

instructor of molecular biology from
the Second

Military Medical University in
Shanghai,

China. Credit: UT
Southwestern Medical

Center.

Although the research indicates it may someday be possible to regenerate neurons from the body's own cells to repair traumatic brain injury or spinal cord damage or to treat conditions such as Alzheimer's disease, the researchers stressed that it is too soon to know whether the neurons created in these initial studies resulted in any functional improvements, a goal for future research.

Spinal cord injuries can lead to an irreversible loss of neurons, and along with scarring, can ultimately lead to impaired motor and sensory functions. Scientists are hopeful that regenerating cells can be an avenue to repair damage, but adult spinal cords have limited ability to produce new neurons. Biomedical scientists have transplanted stem cells to replace neurons, but have faced other hurdles, underscoring the need for new methods of replenishing lost cells.

Scientists in UT Southwestern's Department of Molecular Biology first successfully turned astrocytes – the most common non-neuronal brain cells – into neurons that formed networks in mice. They now successfully turned scar-forming astrocytes in the spinal cords of adult mice into neurons. The latest findings are published today in Nature Communications and follow previous findings published in Nature Cell Biology.

"Our earlier work was the first to clearly show in vivo (in a living animal) that mature astrocytes can be reprogrammed to become functional neurons without the need of cell transplantation. The current study did something similar in the spine, turning scar-forming astrocytes into progenitor cells called neuroblasts that regenerated into neurons," said Dr. Chun-Li Zhang, assistant professor of molecular biology at UT Southwestern and senior author of both studies.

"Astrocytes are abundant and widely distributed both in the brain and in the spinal cord. In response to injury, these cells proliferate and contribute to scar formation. Once a scar has formed, it seals the injured area and creates a mechanical and biochemical barrier to neural regeneration," Dr. Zhang explained.

"Our results indicate that the astrocytes may be ideal targets for in vivo reprogramming."

The scientists' two-step approach first introduces a biological substance that regulates the expression of genes, called a transcription factor, into areas of the brain or spinal cord where that factor is not highly expressed in adult mice. Of 12 transcription factors tested, only SOX2 switched fully differentiated, adult astrocytes to an earlier neuronal precursor, or neuroblast, stage of development, Dr. Zhang said.

In the second step, the researchers gave the mice a drug called valproic acid (VPA) that encouraged the survival of the neuroblasts and their maturation (differentiation) into neurons. VPA has been used to treat epilepsy for more than half a century and also is prescribed to treat bipolar disorder and to prevent migraine headaches, he said.

The current study reports neurogenesis (neuron creation) occurred in the spinal cords of both adult and aged (over one-year old) mice of both sexes, although the response was much weaker in the aged mice, Dr. Zhang said. Researchers now are searching for ways to boost the number and speed of neuron creation. Neuroblasts took four weeks to form and eight weeks to mature into neurons, slower than neurogenesis reported in lab dish experiments, so researchers plan to conduct experiments to determine if the slower pace helps the newly generated neurons properly integrate into their environment.

In the spinal cord study, SOX2-induced mature neurons created from reprogramming of astrocytes persisted for 210 days after the start of the experiment, the longest time the researchers examined, he added.

Because tumour growth is a concern when cells are reprogrammed to an earlier stage of development, the researchers followed the mice in the Nature Cell Biology study for nearly a year to look for signs of tumour formation and reported finding none.

Monday, 24 February 2014

The power of regenerative medicine now allows scientists to transform skin cells into cells that closely resemble heart cells, pancreas cells and even neurons. However, a method to generate cells that are fully mature — a crucial prerequisite for life-saving therapies — has proven far more difficult. But now, scientists at the Gladstone Institutes and the University of California, San Francisco (UCSF), have made an important breakthrough: they have discovered a way to transform skin cells into mature, fully functioning liver cells that flourish on their own, even after being transplanted into laboratory animals modified to mimic liver failure.

In previous studies on liver-cell reprogramming, scientists had difficulty getting stem cell-derived liver cells to survive once being transplanted into existing liver tissue. But the Gladstone-UCSF team figured out a way to solve this problem. Writing in the latest issue of the journal Nature, researchers in the laboratories of Gladstone Senior Investigator Sheng Ding, PhD, and UCSF Associate Professor Holger Willenbring, MD, PhD, reveal a new cellular reprogramming method that transforms human skin cells into liver cells that are virtually indistinguishable from the cells that make up native liver tissue.

These results offer new hope for the millions of people suffering from, or at risk of developing, liver failure — an increasingly common condition that results in progressive and irreversible loss of liver function. At present, the only option is a costly liver transplant. So, scientists have long looked to stem cell technology as a potential alternative. But thus far they have come up largely empty-handed.

"Earlier studies tried to reprogram skin cells back into a pluripotent, stem cell-like state in order to then grow liver cells," explained Dr. Ding, one of the paper's senior authors, who is also a professor of pharmaceutical chemistry at UCSF, with which Gladstone is affiliated.

"However, generating these so-called induced pluripotent stem cells, or iPS cells, and then transforming them into liver cells wasn't always resulting in complete transformation. So we thought that, rather than taking these skin cells all the way back to a pluripotent, stem cell-like state, perhaps we could take them to an intermediate phase."

"Instead of taking the skin cells back to the beginning, we took them only part way, creating endoderm-like cells," added Gladstone and CIRM Postdoctoral Scholar Saiyong Zhu, PhD, one of the paper's lead authors.

"This step allowed us to generate a large reservoir of cells that could more readily be coaxed into becoming liver cells."

Next, the researchers discovered a set of genes and compounds that can transform these cells into functioning liver cells. And after just a few weeks, the team began to notice a transformation.

"The cells began to take on the shape of liver cells, and even started to perform regular liver-cell functions," said UCSF Postdoctoral Scholar Milad Rezvani, MD, the paper's other lead author.

"They weren't fully mature cells yet — but they were on their way."

Now that the team was encouraged by these initial results in a dish, they wanted to see what would happen in an actual liver. So, they transplanted these early-stage liver cells into the livers of mice. Over a period of nine months, the team monitored cell function and growth by measuring levels of liver-specific proteins and genes.

Two months post-transplantation, the team noticed a boost in human liver protein levels in the mice, an indication that the transplanted cells were becoming mature, functional liver cells. Nine months later, cell growth had shown no signs of slowing down. These results indicate that the researchers have found the factors required to successfully regenerate liver tissue.

"Many questions remain, but the fact that these cells can fully mature and grow for months post-transplantation is extremely promising," added Dr. Willenbring, associate director of the UCSF Liver Center and the paper's other senior author.

"In the future, our technique could serve as an alternative for liver-failure patients who don't require full-organ replacement, or who don't have access to a transplant due to limited donor organ availability."

The method also represents an early but important step toward building fully functional replacements for injured or diseased tissue that can be designed from CAT scan data using computer-aided design (CAD), printed in 3D at the push of a button, and used by surgeons to repair or replace damaged tissue.

"This is the foundational step toward creating 3D living tissue," said Jennifer Lewis, Ph.D., senior author of the study, who is a Core Faculty Member of the Wyss Institute for Biologically Inspired Engineering at Harvard University, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard SEAS. Along with lead author David Kolesky, a graduate student in SEAS and the Wyss Institute, her team reported the results February 18 in the journal Advanced Materials.

Tissue engineers have tried for years to produce lab-grown vascularized human tissues robust enough to serve as replacements for damaged human tissue. Others have printed human tissue before, but they have been limited to thin slices of tissue about a third as thick as a dime. When scientists try to print thicker layers of tissue, cells on the interior starve for oxygen and nutrients, and have no good way of removing carbon dioxide and other waste. So they suffocate and die.

Nature gets around this problem by permeating tissue with a network of tiny, thin-walled blood vessels that nourish the tissue and remove waste, so Kolesky and Lewis set out to mimic this key function.

3D printing excels at creating intricately detailed 3D structures, typically from inert materials like plastic or metal. In the past, Lewis and her team have pioneered a broad range of novel inks that solidify into materials with useful electrical and mechanical properties. These inks enable 3D printing to go beyond form to embed functionality.

To print 3D tissue constructs with a predefined pattern, the researchers needed functional inks with useful biological properties, so they developed several "bio-inks" — tissue-friendly inks containing key ingredients of living tissues. One ink contained extracellular matrix, the biological material that knits cells into tissues. A second ink contained both extracellular matrix and living cells.

To create blood vessels, they developed a third ink with an unusual property: it melts as it cools, rather than as it warms. This allowed the scientists to first print an interconnected network of filaments, then melt them by chilling the material and suction the liquid out to create a network of hollow tubes, or vessels.

The Harvard team then road-tested the method to assess its power and versatility. They printed 3D tissue constructs with a variety of architectures, culminating in an intricately patterned construct containing blood vessels and three different types of cells – a structure approaching the complexity of solid tissues.

Moreover, when they injected human endothelial cells into the vascular network, those cells regrew the blood-vessel lining. Keeping cells alive and growing in the tissue construct represents an important step toward printing human tissues.

"Ideally, we want biology to do as much of the job of as possible," Lewis said.

Lewis and her team are now focused on creating functional 3D tissues that are realistic enough to screen drugs for safety and effectiveness.

"That's where the immediate potential for impact is," Lewis said.

Scientists could also use the printed tissue constructs to shed light on activities of living tissue that require complex architecture, such as wound healing, blood vessel growth, or tumour development.

"Tissue engineers have been waiting for a method like this," said Don Ingber, M.D., Ph.D., Wyss Institute Founding Director.

"The ability to form functional vascular networks in 3D tissues before they are implanted not only enables thicker tissues to be formed, it also raises the possibility of surgically connecting these networks to the natural vasculature to promote immediate perfusion of the implanted tissue, which should greatly increase their engraftment and survival".

Researchers at the University of California, San Diego School of Medicine have discovered that a well-known protein has a new function: It acts in a biological circuit to determine whether an immature neural cell remains in a stem-like state or proceeds to become a functional neuron.

The findings, published in the February 13 online issue of Cell Reports, more fully illuminate a fundamental but still poorly understood cellular act – and may have significant implications for future development of new therapies for specific neurological disorders, including autism and schizophrenia.

Postdoctoral fellow Chih-Hong Lou, working with principal investigator Miles F. Wilkinson, PhD, professor in the Department of Reproductive Medicine and a member of the UC San Diego Institute for Genomic Medicine, and other colleagues, discovered that this critical biological decision is controlled by UPF1, a protein essential for the nonsense-mediated RNA decay (NMD) pathway.

NMD was previously established to have two broad roles. First, it is a quality control mechanism used by cells to eliminate faulty messenger RNA (mRNA) – molecules that help transcribe genetic information into the construction of proteins essential to life. Second, it degrades a specific group of normal mRNAs. The latter function of NMD has been hypothesized to be physiologically important, but until now it had not been clear whether this is the case.

Wilkinson and colleagues discovered that in concert with a special class of RNAs called microRNA, UPF1 acts as a molecular switch to determine when immature (non-functional) neural cells differentiate into non-dividing (functional) neurons. Specifically, UPF1 triggers the decay of a particular mRNA that encodes for a protein in the TGF-b signalling pathway that promotes neural differentiation. By degrading that mRNA, the encoded protein fails to be produced and neural differentiation is prevented. Thus, Lou and colleagues identified for the first time a molecular circuit in which NMD acts to drive a normal biological response.

NMD also promotes the decay of mRNAs encoding proliferation inhibitors, which Wilkinson said may explain why NMD stimulates the proliferative state characteristic of stem cells.

"There are many potential clinical ramifications for these findings," Wilkinson said.

"One is that by promoting the stem-like state, NMD may be useful for reprogramming differentiated cells into stem cells more efficiently.”

"Another implication follows from the finding that NMD is vital to the normal development of the brain in diverse species, including humans. Humans with deficiencies in NMD have intellectual disability and often also have schizophrenia and autism. Therapies to enhance NMD in affected individuals could be useful in restoring the correct balance of stem cells and differentiated neurons and thereby help restore normal brain function."

Tuesday, 11 February 2014

New Live-cell Printing Technology Works like Ancient Chinese Woodblock Printing

Monday, 10 February 2014

With a nod to 3rd century Chinese woodblock printing and children's rubber stamp toys, researchers in Houston have developed a way to print living cells onto any surface, in virtually any shape. Unlike recent, similar work using inkjet printing approaches, almost all cells survive the process, scientists report in this week's Proceedings of the National Academy of Sciences.

This
image shows cells printed in a grid pattern

by
block cell printing technology (left) and

woodblocks
used in ancient Chinese printing

(right).
Credit: Lidong Qin lab and Digital

Museum of Science and Art (Beijing, China).

The researchers, led by Houston Methodist Research Institute nano-medicine faculty member Lidong Qin, Ph.D., say their approach produces 2-D cell arrays in as little as half an hour, prints the cells as close together as 5 micrometres (most animal cells are 10 to 30 micrometres wide), and allows the use of many different cell types. They've named the technology Block-Cell-Printing, or BloC-Printing.

"We feel the current technologies are inadequate," Qin said.

"Inkjet-based cell printing leaves many of the cells damaged or dead. We wanted to see if we could invent a tool that helps researchers obtain arrays of cells that are alive and still have full activity."

Recent work to print cells in two and three dimensions using electricity-gated inkjet technology have been largely successful, but sometimes only half of the printed cells survive the printing process – a source of frustration for many laboratory scientists.

"Cell printing is used in so many different ways now – for drug development and in studies of tissue regeneration, cell function, and cell-cell communication," Qin said.

"Such things can only be done when cells are alive and active. A survival rate of 50 to 80 percent is typical as cells exit the inkjet nozzles. By comparison, we are seeing close to 100 percent of cells in BloC-Printing survive the printing process."

BloC-Printing manipulates microfluidic physics to guide living cells into hook-like traps in the silicone mold. Cells flow down a column in the mold, past trapped cells to the next available slot, eventually creating a line of cells (in a grid of such lines). The position and spacing of the traps and the shape of the channel navigated by the cells is fully configurable during the mold's creation. When the mold is lifted away, the living cells remain behind, adhering to the growth medium or other substrate, in prescribed formation.

Qin's group tested BloC-Printing for its utility in studying cancerous cells and primary neurons. By arranging metastatic cancer cells in a grid and examining their growth in comparison with a non-metastatic control, the researchers found they could easily characterize the metastatic potential of cancer cells.

"We looked at cancer cells for their protrusion generation capability, which correlates to their malignancy level," Qin said.

"Longer protrusion means more aggressive cancer cells. The measurement may help to diagnose a cancer's stage."

The researchers also printed a grid of brain cells and gave the cells time to form synaptic and autaptic junctions.

"The cell junctions we created may be useful for future neuron signal transduction and axon regeneration studies," Qin said.

"Such work could be helpful in understanding Alzheimer's disease and other neurodegenerative diseases."

While it is too early to predict the market cost of BloC-Printing, Qin said the materials of a single BloC mold cost about $1 (US). After the mold has been fabricated and delivered, a researcher only needs a syringe, a carefully prepared suspension of living cells, a Petri dish, and a steady hand, Qin said. Inkjet cell printers can cost between $10,000 and $200,000.

"BloC-Printing can be combined with molecular printing for many types of drug screening, RNA interference, and molecule-cell interaction studies," he said.

"We believe the technology has big potential."

While the fidelity of BloC-Printing is high, Qin said inkjet printing remains faster, and BloC-Printing cannot yet print multi-layer structures as ink-jetting can.

A cure for type 1 diabetes has long eluded even the top experts. Not because they do not know what must be done — but because the tools did not exist to do it. But now scientists at the Gladstone Institute’s Investigator Sheng Ding, MD, PhD, harnessing the power of regenerative medicine, have developed a technique in animal models that could replenish the very cells destroyed by the disease. The team's findings, published online today in the journal Cell Stem Cell, are an important step towards freeing an entire generation of patients from the life-long injections that characterize this devastating disease.

Type 1 diabetes, which usually manifests during childhood, is caused by the destruction of b-cells, a type of cell that normally resides in the pancreas and produces a hormone called insulin. Without insulin, the body's organs have difficulty absorbing sugars, such as glucose, from the blood. Once a death sentence, the disease can now be managed with regular glucose monitoring and insulin injections. A more permanent solution, however, would be to replace the missing b-cells. But these cells are hard to come by, so researchers have looked towards stem cell technology as a way to make them.

"The power of regenerative medicine is that it can potentially provide an unlimited source of functional, insulin-producing b-cells that can then be transplanted into the patient," said Dr. Ding, who is also a professor at the University of California, San Francisco (UCSF), with which Gladstone is affiliated.

"But previous attempts to produce large quantities of healthy b-cells — and to develop a workable delivery system — have not been entirely successful. So we took a somewhat different approach."

One of the major challenges to generating large quantities of b-cells is that these cells have limited regenerative ability; once they mature it's difficult to make more. So the team decided to go one step backwards in the life cycle of the cell.

The team first collected skin cells, called fibroblasts, from laboratory mice. Then, by treating the fibroblasts with a unique 'cocktail' of molecules and reprogramming factors, they transformed the cells into endoderm-like cells. Endoderm cells are a type of cell found in the early embryo, and which eventually mature into the body's major organs — including the pancreas.

"Using another chemical cocktail, we then transformed these endoderm-like cells into cells that mimicked early pancreas-like cells, which we called PPLC's," said Gladstone Postdoctoral Scholar Ke Li, PhD, the paper's lead author.

"Our initial goal was to see whether we could coax these PPLC's to mature into cells that, like b-cells, respond to the correct chemical signals and — most importantly — secrete insulin. And our initial experiments, performed in a petri dish, revealed that they did."

The research team then wanted to see whether the same would occur in live animal models. So they transplanted PPLC's into mice modified to have hyperglycaemia (high glucose levels), a key indicator of diabetes.

"Importantly, just one week post-transplant, the animals' glucose levels started to decrease gradually approaching normal levels," continued Dr. Li.

"And when we removed the transplanted cells, we saw an immediate glucose spike, revealing a direct link between the transplantation of the PPLC's and reduced hyperglycaemia."

But it was when the team tested the mice eight weeks post-transplant that they saw more dramatic changes: the PPLC's had given rise to fully functional, insulin-secreting b-cells.

"These results not only highlight the power of small molecules in cellular reprogramming, they are proof-of-principle that could one day be used as a personalized therapeutic approach in patients," explained Dr. Ding.

"I am particularly excited about the prospect of translating these findings to the human system," said Matthias Hebrok, PhD, one of the study's authors and director of the UCSF Diabetes Center.

"Most immediately, this technology in human cells could significantly advance our understanding of how inherent defects in b-cells result in diabetes, bringing us notably closer to a much-needed cure."

Thursday, 6 February 2014

Higher mammals, such as humans, have markedly larger brains than other mammals. Scientists from the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden recently discovered a new mechanism governing brain stem cell proliferation. It serves to boost the production of neurons during development, thus causing the enlargement of the cerebral cortex – the part of the brain that enables us humans to speak, think and dream. The surprising discovery made by the Dresden-based researchers: two components in the stem cell environment – the extracellular matrix and thyroid hormones – work together with a protein molecule found on the stem cell surface, a so-called integrin. This likely explains why iodine deficiency in pregnant women has disastrous consequences for the unborn child, affecting its brain development adversely – without iodine, no thyroid hormones are produced.

This
image shows stem cells in the cortex of a

mouse
embryo (cell nuclei: blue). Credit: MPI

für
Molecular Cell Biology and Genetics/

D. Stenzel.

"Our study highlights this relationship and provides a potential explanation for the condition neurologists refer to as cretinism", says Wieland Huttner, Director at the Max Planck Institute in Dresden. This neurological disorder severely impairs the mental abilities of a person.

In the course of evolution, certain mammals, notably humans, have developed larger brains than others, and therefore more advanced cognitive abilities. Mice, for example, have brains that are around a thousand times smaller than the human one. In their study, which was conducted in cooperation with the Fritz Lipmann Institute in Jena, the researchers in Dresden wanted to identify factors that determine brain development, and understand how larger brains have evolved.

A cosy bed for brain stem cells

Brain neurons are generated from stem cells called basal progenitors that are able to proliferate in humans, but not in mice. In humans, basal progenitors are surrounded by a special environment, a so-called extracellular matrix (ECM), which is produced by the progenitors themselves. Like a cosy bed, it accommodates the proliferating cells. Mice lack such ECM, which means that they generate fewer neurons and have a smaller brain.

The scientists therefore conducted tests to see whether in mice, basal progenitors start to proliferate if a comparable cell environment is simulated.

"We simulated an extracellular matrix for the brain stem cells using a stimulating antibody. This antibody activates an integrin on the cell surface of basal progenitors and thus stimulates their proliferation", explains Denise Stenzel, who headed the experiments.

Because a requirement of thyroid hormones for proper brain development was previously known, the researchers blocked the production of these hormones in pregnant rats to see if their absence would inhibit basal progenitor proliferation in the embryos. Indeed, fewer progenitors and, consequently, neurons were produced, likely explaining the abnormal brain development in the absence of thyroid hormones. When the action of these hormones on the integrin was blocked, the ECM-simulating antibody alone was no longer able to induce basal progenitor proliferation.

"That is probably how, in the course of evolution, we humans developed larger brains", says Wieland Huttner, summing up the study.

The research produced another important finding too.

"We were able to explain the role of iodine in embryonic brain development at the cellular level", says Denise Stenzel. Iodine is essential for the production of thyroid hormones, and an iodine deficiency in pregnant women is known to have adverse effects on the brain development of the unborn child.